CA1315232C

CA1315232C - Cell culture apparatus

Info

Publication number: CA1315232C
Application number: CA000607857A
Authority: CA
Inventors: Bruce P. Amiot
Original assignee: Endotronics Inc
Current assignee: Endotronics Inc
Priority date: 1988-08-10
Filing date: 1989-08-09
Publication date: 1993-03-30
Anticipated expiration: 2010-03-30
Also published as: EP0356785A1; KR970008216B1; US5079168A; JPH02107183A; AU3924089A; EP0356785B1; ATE90723T1; AU622921B2; KR900003359A; DE68907153D1; DE68907153T2

Abstract

ABSTRACT OF THE DISCLOSURE

A compact, easily assembled cell culturing device comprising at least one envelope, the interior of which defines a cell culturing space wherein the envelope is spirally wrapped about an elongated core and which provides for optimal gas delivery and removal to and from the cell culturing space which is also separate from nutrient media delivery and removal whereby greater amounts of oxygen are provided to the cells at a faster rate to produce cells and/or cell products more economically and in higher yield.

Description

This invention relates to a device for cultivating cells in vitro. More particularly, it relates to a compact, easily assembled cell culturing d~vice comprising at least one envelope, the interior of which defines a cell culturing space wherein the envelope is spirally wrapped about an elongated core. The invention relates even more particularly to a cell culturing device, simple in design, yet which provides for efficient gas delivery and removal to and from the cell culturing space to be separate from nutrient media delivery and removal whereby greater a~ounts of oxygen are provided to the cells at a faster rate to produce cells and/or cell products more economically and in higher yield.
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The culturing of living cells in vitro is performed for a variety of purposes including the preparation of vixal v vaccines, the recovery of valuable by-products of cell metabolism and the production of tissu~-like derivatives for creating artificial organs.

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Several problems are as~ociated with growing livlng cell~ in vitro to produc2 dense mas~es of cells. Fir~t, individual component.q of th~ nutrient medium mu~t difu~e through th~ cell layers to reach all cell~. Thig becomes increasingly difficult a3 the thic~nes~ of the cell layer increases.
Second, the maintenance of a sultable environment for cell growth 1~ dlfflcult becaus~ the fluid immediately ad~acent a growing cell i~
continuously changing as cellular m~taboli~m proceed~
and iq returned to its origlnal ~tatus only in ~tepwi~e fa~hion when the nutri~nt medium i8 changed or agitated en masse.
Third, a lattice or ~uitable material upon which to grow some type~ of cells i~ required.
Various types of apparatu~ and methods have been developed in respon~e to these naeds. One method involve~ attaching a~d ~rowin~ cells on the interior ~urface of plastic or gla~s roller tubes and bottles a~ disclo~ed in U.S. Patent No. 3,450,598. Another method involves attaching the cells to a flat ~urface of s~ationary container3 such a~ petri dishe3 or rectangularly shaped culture plate~. The flat surfaces can be stacXed one o~ top of each other in a spaced-apart array a~ disclo~ed in U.S. 3,843,454.

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The use of hollow fibers or synthetic capillaries ha~ more recently been discloged a~ a support matrix for the propagation of cell~. For example, U.S. Patent No~. 3,821,087; 3,883,393:
4,184,922; 4,200,689; 4,206,015 and 4,220,725, all to Knazek et al, variously dl~close apparatua and method~ for the in vitro growth of cell~ on semi-permeable. tubular membranes or capill~ries wherein cell3 are initially allowed to settle onto `` the outer surface~ of the capillary wall~ in a nutrient medium. Nutrients diffuse from the perfuYing medium through the capillary walls and are utilized by tha cells. Cell products diff~s~ from the cell8, through the capillary wall~ and lnto th~
perfusate, from which cell products may be recovered.
U.~. 4,184,922 and 4,200,689 disclose cell culturing device3 comprising a ~ingle bundle of fibers wherein ~ome o the ~lbers are connected to one perfusion circuit and the remaining fibers are connected to a ~econd perusion clrcuit. The difference in pressure between the two c~rcuit~
produce~ convective currents of perfu~ate wi~hin the ; extracaplllary ~pace and thereby impro~e~ nutrlent ~ distrib~tlon to the growing cells.

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In U.S. Patent No. 4,220,725, a bundle of capillarie~, upon wh~ch cell~ are allowed to grow, i~ --wrapped in a porou~ envelope or ~heet material whlch creates an extra-envelope space into which the cell~
can migrate for periodlc removal without dlsturbing the main cell culture. The creation of the extra-envelope space lncrea~es the ~urface ar~a for nutrient end waste product diffuslon to and from the cells located on the outer ~ur~ace of the capi l laries .
In V.S. Patent No. 3,997,396, cells are attached to and grown on one ~ide or surface of a ~ingle hollow iber membrane wherein the cells are propagated and maintalned by passing o~ygen through the membrane from the side oppo~ite that to which the cells are attached and into contact with the cell~
while simultaneously incubating the c~118 ~n a nutrient med~um. By continuou~ly pass~ng oxyge through the membrane from the ~lde oppo~ite that on which the cell~ are attached, a cont~nuous and unifo_m supply of oxygen reache~ and nouri~hes the cell~ thereby facilitating aerobic propagation of tbe cells in the desired ti3~ue denYltie8.
In U.S. Patent No~. 4,087,327 and 4,201,~345 to Feder et al, an in vitro cell culture reaction ~ystem -- ~ 3 ~

i~ disclosed which utilizes elongate hollow or ~olid fibers arranqed in a shallow_layer ~onfiguration a~ a matrix for cell attachment on the outer surface of the fibers. Nutrient media flow i~ directed ~ubstantially uniormly through the fiber layer and substantially tran~ver~e to th~ plane of the elongate axes of the fiber~. The cella are aerated by paq~ing oxygen through the interior of the fibers which then permeate~ the fiber wall~. The use of a ~hallow bed of ibers in a relatively short path of media flo~
re~ult~ in a ~ub~ta~tial reduction of the nutrient and metabolic product gradient~ that i~ normally produced by the fibrous bundle as well a3 a more extensive ut~lization of ~he fiber surface for cell attachment.
U.S. Patent No. 4,391,912 di~closes a device for cultivating floating anlmal cells comprlsing a ga~
permeable shell and a plurality of hollow fibers enclosed within the shell, wherein the hollow fibers are open at either end out~ide of the shell and have a pore diameter of from about 102 ang~troms to 5 x 104 angstroms. Nutrient medium passes through the interior of the hollow flber~ and oxygen passe~
through the shell and the animal cell~ are cultivatsd in the .space bst~een the shell and the hollow fiber~.

These pore diameters of the hollow fiber~ are di clo~ed a~ optimizing efficient exchange o nutrients and metabolic product~ produced by the cells re~ulting in high den~lty cell growth.
Notwlthstanding the usefulnes~ of the hollow fiber cell culture devices, it has been found that the nutrient media flow through the hollow capillarieq prevent~ complete penetration of the capillary bundle by the cell~ and set~ up an unde~irable gradient of m~dlum flow. A~ a re~ult, there i8 an incomplete utilization of the avallable capillary ~urface for cell attac~ment~ and cells become unevenly distrlbuted alonq the ~urface. Also, as the nutrient medium flows ~hrough the reactor, nutrient3 are more available to the cells near the inlet, and a~ the medium flow~ t~ t~e outlet, metabolic products such a~ lactic acid accumulate in the medium, undesirably aff~cting pH and producing other toxic effect~ on the cell~
Another ~i~nificant difficulty encountered with these hollow fiber-type cell culture devices concern~
the high media circulation rate~ necessary to ~upply adequate oxygen to the cell~. Specifically, aqueou~
nutrient media, equilibrated with air, i~ able to carry 4,5 ml of oxygen p~r liter (37C, 760 mm o .

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Hg). This relative inability of aqueou~ ~olution~ to carry oxyqen causes the rate at which oxygen i8 supplied to the cellY to be the limiting ~tep in in vitro cell growth operations. In order t~ produce high yields of cells and/or cell secreted product~, media circulation rates must be increased to provide more oxygen to cells. High circulatlon rate3 ln turn cause high internal pre~sure and turbulence whlch has presented problems in term~ of con~tructlng the device on an indu~trial scale and in propa~ating mammalian cells whose sen~itivity and fragility prohibit the use of too vigorous aeration and/or aqitation. Vigorou~ aeration also causes the denaturation of many ~iologically and medicinally useful proteins produced by cell culture~.
Moreover, the above-described hollow fiber-type device~ which provide for separate oxygen and nutrient media deliYery to cell~ ~uffer from th~
additional disadvantage~ of bein~ mechanically complex, difficult to assemble and being unduly large~ Moreover, the dimension~ of the~e device~ are not constrained to maintai~ the growing cells in close proximity to the nutrient media 8upply ~ource thus causing undesirable nutrlent gradlents.

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Therefore, it has been desirable to provide new cell ; culturing devices for gxowing cells in vitro, particularly mammalian cells, which overcome tlle various difficulties associated with the prior art devices and produce cells andfor cell secreted products more economically and in higher yields.

The present invention provides a cell culturing device for in vitro cell propagation which features optimally efficient gas exchange between the cells and the external environment achieved by delivering and removing gas to and from the cells separately from nutrient media.

The present invention also provides a cell culturing device which allows for dramatically reduced nutrient media circulation rates, thereby affording greater ease in industrial scale-up.

Further the present invention provides a cell culturing device which is ,.~'.

simple in design, easy to assemble, compact in size and has a gentle internal environment in which to grow cells and recover cells and/or valuable cell products in high yields.

. 5 In one aspect the present invention provides in a cell culture device in one embodiment comprising:
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(a) at least one envelope having first and second external surfaces wherein the envelope comprises a first membrane layer sealed to a second membrane layer along their : first and second lateral and longitudinal edges, ~ respectively, to define a cell culturing space therebetween;

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~; (b) a first delivery means for delivering nutrient media 15 to the cell culturing space, (c) a second delivery means for delivering cells to the cell culturing space;

(d) a third delivery means for delivering gas to the cell culturing space;

(e) an elongated core having a longitudinal axis, to which a first lateral edge of the envelope is attached parallel to the axis, and about which the envelope is spirally wrapped, such that the longitudinal edges are _ g _ .

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di~posed in two planes perpendlcular to the longitudinal axi~ of the core;
(f) an adhe~ive means disposed alony the entire length of both of the longitudinal edges of the f i r~t external surf ace of the at lea~t one envelope such that when the envelop~ i3 spirally wrapped about the core, the longitudinal edgee of the fir~t ext~rnal surface fixedly adhere to the core and then to the 3econd external ~urface of a previously wound layer of en~elope whereby a spirally extendin~
intar-envelope gas ~pace iQ cr~ated therebetween;
(g) a flr t removal mean3 for removin~
metabolic waste product~ from the cell culturing space;
(h) a second removal means for removlng cells and/or cell products from the cell culturing space;
(i) a third removal mean~ for removing ~aseou~ wa~te product~ from the cell culturing space;
(~ a first end header means disposed ad~acent to a fir~ end of the core and having ~l 3 ~ r~J ~
inlet means in communication with the delivery means; and (k) a second Qnd header means disposed a~jacent to a second end Of the core and having outlet m~ans in co~munication with at least one of the removal means.

The advantages and ~eatures of the present invention will be more fully understood from the following description of the preferred embodiments, taken in conjunction with the accompanying drawings, wherein:

FIG. I is a perspective view of the cell culturing device of the present invention in assembled condition according to a first embodiment;

FIG. 2 is a cross-sectional view taken along the line 2-2 in FIG. l;

FIG. 3 is an exploded perspective view of a first embodiment of the present invention in its unassembled state;

FIG. 4 is a longitudinal sectional view of an assembled device according to a first embodiment of the present invention;
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FIG. 5 is an exploded perspective view of a second embodiment of the present invention in itsunassembled state;

FIG. 6 is a longitudinal sectional view of an assembled device according to a second embodiment of the present invention;

FIG. 7 is a cross-sectional view similar to the cross-sectional view of FIG. 2, but showing the arrangement for the second embodiment;

FIG. 8 is a detailed perspective view of a third embodiment of the present invention;

FIG~ 9 is an exploded perspective view of a third embodiment of the present invention in its unassembled stake;

FIG~ 10 is a cross-sectional view similar to FIGo 2 but showing the arrangement for the third embodiment; and ~0 FIG. 11 is a longitudinal sectional view of an assembled device according ts a third embodiment of the present invention.

In the various embodiments of the invention as illustrated in the drawings, like structures will be referred to by like reference numerals.

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In the embodiment of the invention as ahown in FI~. l, an assembled cell culturing device i3 provided as generally indicated at 12. In thi3 embodimentt the cell culturing devlce 12 includes an outer shell or jacket 14 which i~ prefera~ly an ela~tomeric sleeve made of 8il~ cone or a heat shrinkable thermoplastic sleeve, the surface o which has perforations 15. Shell 14 and the end cap headers 60 and 70 together form an exterior enclo~ure of the cell culturing device 12.
FIG. 2 i8 a cros~-sect~onal view of the fir~t embodiment of the inventlon as ill~trated ln FIG. 1.
The speclfic feature~ of thia figure will be described upon reference to FIG. 3, descrlbed in detail hereinafter.
Turning now to FIG. 3, which illustrates a fir~t embodiment of the present ~n~ention in it~
unassembled ~tate, a cell culturing envelope 20, having first and second external surfaces which are deined by ~irst and 6econd membrane layer~ 22 and 24, respectively, having sub~tantially equal dimensions and which are 3ealed to each other along their firat and second longitudinal edgea and flr~t and second lateral edge~, re~pectively, wi~h a suitable adhe~ive 21 to define a cell culturing 3pace 14 ~. 3 ~ 3 ~

26 therebetween. Silico~ adhesives are preferr~d.
The layer~ ~2 and 24 are preferably made of a porou~
hydrophobic material ~uch a~ medical-grade ~illcone, micro-porou~ polyethylen~, poly~ulfone, polycarbonat@
or polyethylene which i~ permeable to ga~es Ruch as air, oxygen and carbon dioxide but impermeable to cells and liquid3. The porou~ membr~ne layer~
generally have a pore ~ise ln the range rom abo~t 0.02 to 0.4 micron~. A preferred material i~
medical-grade silicone becau~e it does not require additional chemical or phy~i~al modificatlon~ of it~
~urface to permit the effective attachment of cell~
thereto and provide~ for opti~ally effic~ent ~a~
exchange ~etween the cell cultur~ng space 26 and ~h~
external environment. A particularly preferred material i8 a fabric-reinforced polymethyl~iloxane produced by SciMed Life System~ of Mlnneapoll~, MN
and by Dow Coxning of Midland, MI under the ~a~
"~ILASTIC". Membrane layers 22 and 24 should also be constructQd to be as thin as possible to minlmize their re~istance to gas difusion. For example, when ~ilicone l~ used a3 the membrane material, a ~uitable thicXne~s is about 0.125 mm. A preferred thickneR~
of the layer material i~ in the range of from about O.1 mm to 0.250 mm, The above-mentioned hydrophobic : . .

materials are al~o advantageous ln that they do not allow water films to form on their ~urface which increases resi~tance to gas diffu~ion. The longitudinal len~th of the envelope, relative to an external circumference of the core 50, ~hould allow for a plurality of layer~ of envelope to be spirally wrapped about the core. In practice, the longitudinal length of the envelope may be from ~bout 1 m to 90 m, preerably from about ~ m to 40 m and its lateral length or width i~ generally in the range from about 0.1 m to about 0.3 m, and prefarably from about 0.15 m to about 0.25 m.
In this embodiment, nutrient media i8 delivered to and water-soluble waste products are remov~d from the cell culturing space by a plurality of hollow fibers or cap~llaries 28 having liqu~d permeable walls, wh~ch are disposed withln the envelope and whose open end~ extend outwardly from between the sealed longitudinal ed~es of the envelope ~uch that the capillaries communicate with the cell culturing space only through the walls o the capillarie~. The capillaries can be dispo~ed in the envelope ~ub~tantially equ~lly ~paced from and parallel to each other. The distance between the cap~llarie3 i~
generally ~rom about 100 micron~ to about 1000 1~ .
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micron~; the preferred distance i9 from about 200 microns to about 500 micron~. A di~tanc~ of 123~
than 100 microns i~ difficult from a man~fa~turing point of view and does not allow adequate space for cell growth. Spacing o more than 1000 micron~ tend~
to cause nutrient gradient~ to develop ln the cell culturing ~pace which, a~ discussed a~ove, resulte in le~ than optimal cell and~or cell product y~ld.
The cap~llarle~ can be produced from any suitable material which is non-toxic to cells and which can be spun into fibers which for~ a ~emi-porou~, hydroph~lic, and selectively permeablo membrane wall.
Examples include cellulose acetat~, anisotroplc polysulfone, polyethersulfonc, oaponlfled c~llulo~e ester, nylon, polyacrylonitrile and acrylic `.copolymer~. A preferred material is "CUPROPHAN~, ~
regenerated cellulose acetate manufactured by Enka Ltd., Del Ray, C~. The capillarie3 28 transport fresh nutrient media containing glucose, amino acids, vitamin~ and other e~ential nutrients nacessary for speciic cell metabolism requirements to the cell~. The medla dlffuse3 through the capillary walls lnto the cell culturing space 26.
Callular waste product~ diffuse fro~ the cell cult~ring ~pace 26 into the capillarie0 28 and are ~ra~ l~ctrk - 17 - ~ t'`.,;~ ~

carried a~ay by the media flowing therethrough. The external diameter ~f the-capillarie~ 28 i~ generally in the range from about 60 to 400 microns, preferably from about 200 microns to 300 microns; the lnternal diameter is generally from about 100 to 300 microns, preferably from about 200 microns to about 250 micron~.
Acce~s into the cell culturing space 26 for both the delivery of cells and the removal of cell~ and/or cell product~ i~ provided by entry and exit tubes 30 and 32, respectively. Tube 30 is di~posed between the fir~t lon~itudinal edges of tha layers 22 and 24 of the envelope and projects into the cell culturing space such that a fir3t end portion of the tube communicates with the cell culturing space and a second end port~on of the tube communicate~ with extra-capillary lnlet port 68 on the f~r~t end cap header ~0 a~ illustrated in EIG. 4 di~cussed below.
Tube 32 is disposed between the second longitudinal edges of the layer~ 22 and 24 of the envelope oppo~ite the edges whi~h tube 30 i3 disposed such that tube 32 pro~ect~ ~nto the cell culturlng space whereby a fir~t end portion of the tube communicate~
with the cell culturing space and a second end port~on of the tube communicates with extra-capillary outlet port 78 on the se~ond end cap header 70 as illustrated in FIG. 4. The entr~f and exit tubes may be con~tructed of a flexible biocompatible material such as Rilicone rubber, polyethylene or polyurethane. A preferred material ls ~ilicone rubber. The internal diameter of the tubes ~hould be sufficiently large to allow for adeguate cell inoculation into the cell culturing ~pace and th~
removal of cells and~or cell product~ therefrom. A
suitable internal dlameter i5 generally in the range from about 1.5 mm to about 9.S mm. The e~ternal diameter of the tube~ 18 ~elected to facilltat~
bonding or sealing of the tube~ 30 and 32 between ~he longitudinal edge~ of membrane layer~ 22 and 24 which comprise en~elope 20. A preferred external diameter is about 3 mm to about 13 mm. Tubes 30 and 32 extend far enough into the cell culturing ~pace 26 ~erely to provide for adeguate cell inoculation and cell and/or cell product harve~t~ng, respectively. Altho~gh not shown in FI~. 3, more tha~ one of each of tube~ 30 and 32 may be appropriately disposed in the envelope.
Upon aRsembly of envelope 20 having a plurality of capillarie~ 28 and at least one of each of t~be~
30 and 32 dispo~ed therein, an adhesive 21 is applied along the entire length of both longitudinal edges - 19 3 C3~ 3 j~

and alon~ a first lateral edge 27 of a fir~t external surface of the envelope. Then, a ~upport me~h 23 in the form of a sheet having dimen~lons ~uch that its longitudinal and lateral edgea are substantially close to but do not contact the adhesive 21 i~
superimpo~ed on the ir~t external qurface of the envelope. The mesh preferably compr~se~ a non-wov~n pla~tic screen having a thickness of from about 0.5 mm to about l.0 mm. Next, as illu~trated in Fig. 3, the irst lateral edge 27 i~ adhesively attached to an elongated core 50 parallel to the longitudinal axis of the core. Envelope 20 i9 then apir~lly wrapped about the core such that the longltudinal edges of the envelope are disposed in two plane~
perpendicular to the longitudinal axis of the core and ~uch that the longitudinal edgeq of the first external surface of the envelope fixedly adhere to the core and, once having completely covered the external circumferential surface of the core with one wrap, fixedly adhere to the longitudinal edges of a second external ~urface of the previou~ly wrapped layer of envelope. The envelope, when ~pirally wrapped about the core in thls manner, forms a spirally extending inter-envelope space 29 having the mesh 23 contained therein, a~ strated in FIG. 2, . .. .

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described in detail below. Inter-envelope space 29 allows or the unre~tricted and spirally outward flow of gaseous wa~te products which have diffused thereinto fr~m the cell culturin~ spac~ through membrane layers 22 and 24. The mesh function~ to maintain the inter-envelope space by prevent1ng adjacently wrapped layers of envelope from cominy into contact with each other as cell culturing ~pace 26 becomes filled with nutrient media and the cell density increases. The inter-envelope space can be maintained in alternatlve ways be~ides the u~s of the me~h as will be apparent to one of ordinary skill in the art.
The elongated core 50 in thi~ embodiment of the invention ~erve~ a dual purpose. First, the core unctions a~ a ~upport for the splrally wrapped envelope. Second, the core ~erves as a ga~ permeable conduit through which ga~ i8 allowed to fl~w along the entire longitudinal length of the core and diffuse radially outward therefrom. Therefore~ the core i~ ~uitably made of a material sufficient rigid to prov~de support for the envelope as well as ~eing ~ufficlently porou~ to permit the flow of ga~
therethrough ~uch a~ a plastic, metal or alloy. A~
illustrated ln F1g. 3, tho core can be madc o .

~ ` ~ ' `' , . , h ,~i hollow, rigid material whose surface i~ perforated with opening~ or pores_52 to permit the diffusio~ of ga~ thererom. The pore ~ize ~s generally in the range of from about 0.5 mm to 5 mm and preferably from about 1.25 mm to 2.50 mm. Although not show~ in the drawing~, the core ca~ alternatively be made of a porous solid material s~ch a~ microporoua polyethylene, ceramic or other slntered materi~l.
After the envelope i~ completely wrapped about the core, the wrapped sub-assembly can b~ optionally fitted with a ~hell or ~acket 14 such a~ a heat shrinkable thermoplastic leeve a~ illustrated in Figs. 1 and 2. S~itable ex~mple~ of matorials which can be uQed ~nclude PVC (polyvinylchloride), polyolefin, TEFLON, MYLAR~ polyethylene and KYN
~polyvinylidene fluoride). Of cours~, ln thi~
embodiment, if a protectivo sh~ provided a~
show~, it mu~t either be ga~ permeable and~or perforated (as ~own in Fig~. 1 and 2) to allow for the e~cape of gas from the ~evlce. A second function of th~ 3hell or ~acket iB to con~train the spirally wrapped envelope, ~o that once filled with a nutrient medium, the cell culture chamber located be~ween the sheets 22 and 24 Will have a total ~hicXnes~ of no greater than from about 200 to about 500 micron~, ~rc~de- ma~k .. .. .

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preferably no greater than from about 200 to 350 micron Once the envelope i9 wrapped about the core and encased within a shell in the above-described mann~r, a first end cap header 60 1Y di~posed adjacent a fir~t end of the core as lllu~trated in Fig~ 4.
Annular shoulder or flange 62 of header 60 iB f~xedly attached to the external circumerential edge of shell 14 to form a hydraulic seal therewlth.
Extra-caplllary inlet port 68 i~ adapted to receive cell inoculation tube 30 ~o as to be in fluid communication therewith. Gas inlet port 66 i~
attached to a pump or a compressed ga~ apparatu~
external to the dev~ce for the supply of air to the device and i~ adapted at the interior thereof to engage a first end of the core to be in fluid communication therewith ~nd provide for the unre~tricted flow of a gae ~uch as air into the core.
In this embodiment, an auxlliary header 67 is adapted to gas inlet port 66 and who~e annular shoulder 65 engage8 the open end of core~50 so as to be in fluid communication therewith. A~ used here~n, the tenm "air" is con~trued -to mean not only atmo~pheric air but also gase~ and mixture~ thereof which are non-toxic and physiologlcally accept~le to cell~ and - 23 - ~ 3 ~ J ~

which are conducive to their grow~h in vitro.
:~ Alternatively, auxiliary header 67 can be replaced by a plastic tube which is adapted at it~ flr~t and second ends to be in communicat~on with the ga~ inlet port and the open end o the core, re~pectively.
Intra-capillary inlet port 64 i~ attached to an external nutrient media ~upply source and i8 adapted at the interior of the devlce to communicate with plenum chamber 63 which i8 de~ned ~y end cap header 60, auxiliary header 67 and the pl~ne de~ined by the ; ~urface of core 50 perpendicular to ite longitudinal axis and the first longitudlnal edge~ of each succes~ively wrapped layer of envelope which are sealed to each other. Plenum chamber 63 comm~nicate~
with fir~t open ends of capillaries 28 which extend outwardly from the ~ealed longitudinal edge~ of the envelope and into the chamber. It i~ preferred that the capillaries are cut f lu3h with the above-descrlbed plane.
A second end cap header 70 is di~po~ed ad~acent a second end of the core such that annular shoulder or flange 72 of header 70 fixedly attachee to the external circumferential edge of the shell 14 ~o form a hydraulic seal therewith. Of course, lf a ~hell were not provided, annular ~houlder~ or ilanges 62 ' : .
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- 2~ -and 72 would be ixedly attached to the external circumferential edge of the outermo~t ~pirally wrapped layer of envelope. Extra-caplllary outlet port 78 i~ adapted at the interior of the device to ~eceive product harve~t tube 32 for the removal of cells and/or cell products from cell culturing spac~
26. Plug 77, attached to header 709 engages the interior circumferential surface of core 50 to prevent the e~cape of gas therefrom. Of cour~e, plug 77 need not be attached to hea~er 70 but ean be supplied separately. Alternatively, a suitable cap can be fitted onto the end of core 50 in a fluid-tight fa~hion or the core can be constructed to have a sealed second end. Intra-capillary outlet port 7~ is adapted at the interlor of the device to communicate with plenum chamber 79 wh~ch is defined by header 70, plug 77 and the plane defined by the surface of core 50 perpendicular to it~ longitudinal axis and the second longltudinal edges of each successively wrapped layer of envelope which are sealed to each other, and into which the second open end~ of capillarie 2B extend.
Fresh nutrient media i~ cau~ed to flow in~o the device through intra-capillary inlet port 64 into plenum chamber 63 which hen perfuses through the . - 25 -u~ ~ 2 capillaries via the first oper, ends thereof. Media and nutrient~ difuse through the capillary walls into cell culturing space 26 and are taken up by the cells. Used media and water-soluble cellular metabolic waste product~ then diffuse from cell culturing ~pace 26 through the capillary walls and are carried by the perfusing media from capillarie~
28 into plenum chamber 79 and out of the device through intra-capillary outlet port 74.
Gase~ are ~upplied to and removed from the device in the following manner. As illustrated in Fig. 4, air 1~ caused to flow lnto the device ~hrough gas inlet port 66 and auxiliary header 67 into core 50. Turning now to Fig. 2, air flow~ through pore~
52 into inter-envelope ~pace 29 and flows spirally therethrough and diffuses through membrane layer~ 22 and 24 into cell culturing space 2~ and is taken up by the cell~. Gaseou~ waste products diffuse throu~h membrane layers 22 and 24 into inter-envelope space 29 and are ca~sed to flow spirally therethrough and outward from core 50 through a space defined by the second lateral edge 25 of envelope and the previou~ly wrapped` layer which are unsealed. Gas exit~ the davice by flowing through opening3 15 on 3hell or ~acket 14.

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A second embodiment of the invention is illustrated in Figs. 5-7. This embodiment differ~
from the first embodiment in several respect~, all of which relate to the way ln which ga~es are ~upplied to and removed from the growing cell~ in the cell culturing pace.
In Fig. 5, envelope 20 having dispo3ed there~n capillarie~ 28 and tubes 30 and 32 is a~sembled a~
described above. Next, adhe~ive 21 i9 applled along the entire length of the fir~t and second longitudinal and lateral edges of the first external surface of envelope 20. Then, at least one qa~ inl~t tube 40 i~ dispo~ed on and secured to the flr~t external surface of envelope 20 and which extend~
along at least a portion of the 3urface thereof so a~
to be parallel with the longitudinal axi~ of core 5~
and which al30 extends outwardly from th~ envelope such that when the envelope i3 spirally wrapped about the core and the device i~ fully a~sembled, one open end of tube 40 i3 easily adapted to communicate with gas inlet port 66' on end cap header 60' and whose other open end communicate~ with inter-envelope gas space ~9, illu3trated in Flg. 7, discussed below.
Disposed on and secured to the oppo~lte longltudinal edge of the fir~t external ~urface of envelope 20 i~

, ,' ' ~ ., ' -~ - 27 -~ 3 ~ J

at least one gas outlet t~be 42 which also extend along at least a portion of th~ ~urface thereof and which also extend6 outwardly from the envelope ~uch that when the envelope i8 spirally wr~pped about the core and the device i8 fully as~embl~d, one open en~
of tube 42 ~ easily adapted to communicate with th~
ga~ outlet port 76' on header 70 and the oth2r open end communicates with inter-envelope gas space 29.
Me~h 23 i8 then superlmpo3ed on the first external ~urface of envelop~ 20 ln the ~ame manner as described above. Fir9t lateral edge 27 of envelope i ~ adhe~i~ely attached to core 54. The core in this embodiment 15 impermeabl~ to gase~ and can be comprised of any matertal which accompli~he~ thi~
purpose and which al~o provide~ support for the spirally wrapped envelop~. Envelope 20 i8 then spirally wrapped ln the same manner a~ descr1bed above except that upon the Gompletion of the wrappin~ the second lateral edge 25 of envelope ~0 havlng adhe9ive 21 ~upplied thereon adheres to the second external ~urface of the previously wrapped layer of envelope. Thi~ wrapped assembly can then be optionally fitted wlth a ~hell 16, as illu~trated in Fig~. 6 and 7 described below. In thl~ embodlment~
`the ~hell can be a heat ~hr~nXable thermoplastic - 2~ t ~ J

sleeve as in the first embodiment but is different in that it need not be permeable to gases and/or perforated. In this embodiment, the shell insures that :the cells in cell culture space 26 will be no further than from about 100 microns to about 250 microns from an oxygen source which in this embodiment is the space inter-envelope space 29 which is supplied with oxygen by tube 40.
`: Once the ~nvelope 1~ wrapped about tho cor~ a~d enca~ed within ehell 16 ln the above-de~crlb~d manner, a first end cap header 60' i~ disposed adjacent a first end of core 54 a~ llluatrated ~n Fig. 6. Annular ~houlder or flang~ 6~ of he~der 60 ;~ i9 fixedly attached to the external circumferentlal edge of shell 16 to orm a hydraulic seal therewith.
~ Extra-capillary inlet port 68' i8 adapted to receive ;cell inoculation tube 30 to be in fluid communica~ion therewith. Ga~ ~nlet port 66 i9 attached to a pump or a compressed ~as mean- external to the dev~c~ or the supply of alr to the devic~ and i~ adapted at the `.interior o the device to enga~e gas iDlet tube ~0 to be in fluid communication therewlth and prov~de for th~ unrèstricted flow of a ga~ ~uch a~ alr thorethrough. ~ntra-caplllary inlet port 64' 18 attached to a nutrlent ~edia supply sourc~ or re~ervoir external to the device and i8 adapted ~t !~ .

,, ~ ` ' ~ ' " ~ ' .

~ ' , 2 g ~ ~ ~ .J ~

the interior thereof to communicate with plenum chamber 63 which 1Y defined by header 60' and the plane defined by the surface of core 54 perpendt~ular to it~ longitudinal axl3 and the first long~tudinal edges of each ~ucce99ively wrapped layer of envelope which are sealed to each other. Plenum c~amber ~3' communicates with fir~t open end~ o~ the capillaries 28 which extend outwardly from the sealed longitudinal edge~ of the envelope and into the chamber. A sacond end cap header 70 is disposed adjacent a second end of the core such that annular shoulder or flange 72' of header 70' fixedly attache~
to the external circumferential edge of the sh~ll 16 to form a hydraulic seal therewith. Of cours~, a~
per the first embodiment, if a shell were not provided, annular shoulders or flanges 62' and 72' would be fixedly attached to the external circumferential edge~ of the outermo3t ~pirally wrapped layer of envelope. Extra-capillary outlet port 78' i~ adapted at the interior of tha devlce to be in fluid communication with product harve4t tube 32 for the removal of cells and/or product~ ~rom cell culturing space 26. Intra-caplllary outlet port 74' i~ adapted at the lnterior of the devicc to communicate with plenum chamber 79' which i8 defined by header 70 and the plane defined by the surface of core 54 perpendicular to its longitudinal axi~ and the second longitudinal edge~ of each ~uccessively wrapped layer of envelope which are sealed to each other, and into which the ~econd open ends of caplllarie~ 28 extend. Gas outlet port 76 1~
adapted at t~e interior of the devlce to be in 1uid communication with ga~ outlet tube. 42 for the removal of gaseous waste products from the device.
Nutrient media is caused to flow into and out of the device ~ub~tantially in the manner de~cribed above for the first embodiment.
Gase~ are cau~ed to enter and exit the device in the following manner. A~ illu3trated in Eig. 6, air is caused to flow from an air supply source external to the device not shown and into the device through ga~ inlet port 66' and then into gas inlet tube 40.
Turning now to Fig. 7, gas flows from tube 40 into inter-envelope ga~ space 29 and diffuses through mèmbrane layer~ 22 and 24 into cell culturing ~pace ~6 where oxygen is taken up by the cell~. Gaseou~
waste products diffu3e through membrane layers 22 and 24 and lnto lnter-envelope ga~ ~pace 29. Turnin~
back to Fig. 6, gase~ thon ~low through ga~ outlet tube 4~ and exlt the device through ga~ outle~ port -~ 3 ~ I ~ J
76'on header 70' which i~ in fluid communiçatlon with gas ou~let tube 42.
In thi~ embodiment, although only one of each of tubeq 40 and 42 i9 shown, the numb~r of gaa inlet tube~ 40 is de~irably one greater than the number of ~a~ outlet t~bes 42. It is al50 pref~rred that th~y be arranged along oppo~ite longitudinal edges of the first external ~urface of the envelope ln an alternating fash~on. Of cours~, ga~ lnl~t port 66 and ga~ outlet port 76 would be adapted at the interior of the device to engage a plurality of tubes 40 and 42, re3pectively, in a manner known to those ~killed in the art.
Thi 8 embodiment i~ advantageou~ in that an oxygen gradient is not created in the cell culturing space 26 ~uch that cells growin~ in the area of the cell cultur~ng space di~po3ed farthest from the external ~urface of the core are not deprived of the oxygen nece~sary for growth.
In th~ above-described embodiment~ o~ thl3 invention, cell~ can be qrown by initially allowing them to attach to the ~all~ of the hollow fi~er~ and the interior surface~ of the envelope or by allowing them to `'float" between the hollow flber~ as di~closed in U.S. 4,391,912.

~ J,~

A third embodiment of the pre~ent invention i~
illu~t~ated in Figs. 8-11. Thi~ embod~ment i~
similar to the second embodiment; however, it differ~
from the second embodiment in several significant way~. Fir~t, as qhown in Fig. 8, two envelGpes 80 and 90 are provlded. Each envelope i~ formed by superimpo3~ng and bonding a gas-permeable hydrophobic membrane layer 22 and 24 onto a semi-porou8 hydrophilic membrane layer ~2 and 92, respectively, with a suitabl2 adhe3ive 21 placed along all four edges of at lea t one membrane layer, thereby forming cell culture ~pace 85 and 95, respectiYely, therebetween. Sultable material~ for the gas-permeable hydr~phobi~ layer~ include tho~e mentioned above in the de~cription of the fir~t embodiment. Suitable material~ for the semi-poro~s hydrophilic layer~ include~ t~ose used to make the capillaries as described ab~ve. Cell inoculation tube 30 and product harvest tube 32 are di~posed within envelopes 80 and 90 as de~crlbed above or the first two embodiments. It i8 noted that ln thl~
embodiment of the present inventlon, capillarie~ are not used. A me3h 23 a~ descrlbed above is then placed atop membrane layers 22, ~2 7 92 and 24. Then, envelope 80 i3 superimposed on envelope 90 such that .
- '.

.

membrane layer~ 82 and 92 Eace one another and are bonded toqether along both thelr lateral edge~ with a suitable adhe~ive. Their longitudinal e~ge~ remain un~ealed.
Turning now to Fig. 9, a suitable adh~ive i8 applled to fir~t and ~econd longitudinal and lateral edges o the external surface of layer 22. Then, gas inlet tubes 40 and gas outlet tube~ 42 are di~posed on and secured to the external surface of layer 22 as de~cribed above for the second embodiment. A fir~t lateral edge of layer 22 is attached to core 54 along it~ longitudlnal ~urface. The core in thi~
embodiment is Rubstantially the same as de cr~bed for the ~econd embodiment.
Envelope~ 80 and 90, assembled and adhered to each other as de~cribed, are ~pirally wrapped about core 54 where upon completion of the wrapping, the second lateral edge 25 of layer 22 a~heres to the external ~urface of layer 24 of the praviously wrapped layer of envelope. Thls i3 ~llu~trated in Fig. 10. Al~o, spirally extendinq inter-envelope space 89 and ~pirally extending inter-envelope channel 99 are created, e~ch having mesh contained therein for the purpose of maintaining adeguate space or ga~ and media flow, respsctively. Inter-envelope ,, " ~, spaca 89 i~ deflned by the adjacent spirally wrapped layer3 22 and 24 whose lateral and longitudinal edges are sealed to each other. Channel 99 i~ defln~d by adjacent spirally wrapped layer6 82 and 92 who~e longitudinal edges are un~ealed and whose lateral edges are sealed.
The wr~pped sub-a3semb1y can then be optionally fitted with a jac~et or shell 16 and then have flrat and second end cap header~ 60" and 70" engage firs~
and second ends, respectively, of ~hell. I6 as de3cribed above for the 3econd embodiment and a~
illustrated in Fig. 11.
Fresh nutrient media i8 cau~ed to flow lnto the device from an external media supply ~ource t~lrough inlet port 64" and into plenum chamber 63 n which i ~
defined by end cap header 60" and the plane defined by the surface of core 54 perpendlcular to itY
longitudinal ~urface and th~ first longitudinal edge~
of each ~ucces~ively wrapped layer of envelopes sn and 90. Nutrient media then flow~ into channel 99 ~illu~trated in Flg. 10) whic~ i in fluid communication with chamber 63~, between the unsealed ad~acent spirally extendlng longitudlnal edge3 of layers 82 and 92, and dlffu~e~ into cell cultur~
spaces 8~ and 9~ respectively, where lt ls taken up ' .. . .

~ 3 ~
by the cell~. U~ed media and water soluble metabolic waste produc~ diffu e from cell culturing spaces 85 and 9S through m~mbrane layers 82 and 92, respectively, into channel 99 and are carrled by perfu ing media into a ~econd plenum ch~mber 79"
defined by end cap header 70" and the plane deflned by the ~urface of core 54 perpendlcular to ~t~
longitudinal axis and the second longitudinal edge~
of each ~uccessively wrapped layex of envelope3 30 and 90 and which i9 in fluid communicatlon wi~h channel 99. Nutrient media ex~ts the device by flowinq through outlet port 74~ which ~ ln fluid communicat~on with chamber 79".
Gase~ are supplied to and removed from th~
device ~b~tantially as de9cribed for th~ second embodiment and as illu~trated in Figs. 10 and 11. As ~hown in Fig. 11, alr flows into the device through gas inlet port 66" which i9 ~adapted at the Interior o the device to be in fluid communicat~on wlth ga~
lnlet tubes 40. Turning now to ~g. 10, ga~ flow~
through tubes 40 into inter-envelope space 89 and diffu~es into cell culture ~pace~ 85 and 95 ~hrough membran~ layers 22 and 24, respectiv~ly, and i~ taken up by the cells. Ga~eou~ waste products exit cell culturing space~ 85 and 95 by diffu~ing through , " ~, ~
:
membrane layers 22 and 24, reqpectively, and into inter-envelope ~pace 89. Turning back to ~ig. 11, gaqe~ then flow through tubes 42, adapted to be in fluld communication with gas outlet port 76" on end cap header 70'` and exit~ the device therethrough. In Fig. 11, only one o each of tuba~ 40 and 4~ are shown becau~e they are coplanar w~th each other.
Thu8, thi~ embodiment of the pres~nt invention al~o provides or the separate supply and removal of nutrient media and gase~ to and from the cell~ yet without the use of caplllarie~.
Any of the three embodiment~ hereinbefore ; de~cribed can be modified in the follow~ng way.
~4~ To încrease the amount of 3urface area in the cell culturing space for the cells to attach, any ~, po~itively charged non-to~ic part~ cle8 of from about 200 to 400 microns in diameter ~uch a~ microcarrier~, reticula~ed polyurethane foam or ~ilica particles can . be added to the cell culturing space and enclos~d therein upon assembly of the envelope.
~ A wide variety of different types of animal t cell~ can be cultured ln the device of this invention including, for example, amphibian, mammalian and avian cell~, partlcularly mammallan cell~. Example3 thereof Inalude human lung ibroblast cell~, ~he~u~

., t . , .: ' ~, ~ , ' , . - 37 ~

monkey kidney cells, vero cellsl MDC~ cells, Chinese hamster ovary cells, chick fibroblast cells, mouse embryo fibroblast cells and baby hamster kidney cells.
Bacterial cells, insect cells, and plant cells can also be cultured therein but this invention is particularly applicable to culture of animal cells as listed above.
The device is also adapted to be used with any conventional nutri~nt media such as Eagle's basal medium Dul~ecco's modified minimum essential medium (MEM) and Earle's or Hank's balanced salt solutions fortified with appropriate nutrients, fetal calf sera, and other materials.
By providing a gas such as oxyyen to the cells in the manner described according to the present invention, cells can be grown more economically and cells and/or cell products can be produced in higher yields because the rate at which oxygen is delivered to the cells is greatly increased over what i~ is in prior art devices. For example, aqueous nutrient media e~uilibrated with air can carry only 4.5 ml of oxygen per liter of 37C and 760 mm Hg pressure while air under the same conditions can carry 209 ml sf oxy-gen per liter. Thus, at least 46 (209/4.5) ~imes more oxygen is available from a liter of air than a liter of water. Moreover, since a gas such as air is less ~ ." . .

~ 3 ~ y~
vi~cous than water, at any given pres~ure, a greater amount of air per unit time can be delivered to the cell culturing device, thu3 maint~ining a hlgh oxyyen gradient. Therefore, in the pre3ent invention, the increase in cell~ and/or cell pr~duct can be at lea~t 10 to 46 time~ the yield obtained in conventional device~. Also, the fluid flow rate of the non-aerated media can be decreased to be in the order of from 100 to 1000 ~.1 of media per hour per sguar~
meter of cell culturing device. It i~ noted that existing cell culturing devlce~ operate in the order of 500 ml of media per minute per square met~r of device in order to supply -the amount of oxygen neces3ary for cell growth. Thl~ raduction in flow rate and the commensurate reduction in internal pre~sure facilitate indu3trial ~cale-up.
Although the pre~ent invention has been de~cribed in detail and with reference to specific embodiment~, thereof, it will be apparent to one skilled in the art that changes may be mad~ in form and detail without departing from the ~pirit and SCOpQ of the invention.

.

Claims

1. A cell culture device comprising:
(a) at least one envelope having first and second external surfaces wherein the envelope comprises a first membrane layer sealed to a second membrane layer along their first and second lateral and longitudinal edges, respectively, to define a cell culturing space therebetween;
(b) a first delivery means for delivering nutrient media to the cell culturing space;
(c) a second delivery means for delivering cells to the cell culturing space;
(d) a third delivery means for delivering a gas to the cell culturing space;
(e) an elongated core having a longitudinal axis, to which a first lateral edge of the envelope is attached parallel to the axis, and about which the envelope is spirally wrapped, such that said longitudinal edges are disposed in two planes perpendicular to the longitudinal axis of the core;
(f) an adhesive means disposed along the entire length of both of the longitudinal edges of the first external surface of the at least one envelope such that when the envelope is spirally wrapped about the core, the longitudinal edges of the first external surface fixedly adhere to the core and then to the second external surface of a previously wrapped layer of envelope, whereby a spirally extending inter-envelope gas space is created therebetween;

(g) a first removal means for removing liquid metabolic waste products from the cell culturing space;

(h) a second removal means for removing cells or, cell products or a mixture thereof from the cell culturing space;

(i) a third removal means for removing gaseous waste products from the cell culturing space;

(J) a first end header means disposed adjacent to a first end of the core and having inlet means in communication with the delivery means; and (k) a second end header means disposed adjacent to a second end of the core and having outlet means in communication with at least one of the removal means.

2. A cell culture device according to claim 1, wherein said first and second membrane layers comprising said envelope are porous and are substantially permeable to gases but substantially impermeable to cells and liquids.

3. A cell culture device according to claim 2, wherein said membrane layers have a pore size of from about 0.02 to 0.4 microns.

4. A cell culture device according to claim 2, wherein each of said membrane layers comprise medical-grade silicone,

5. A cell culture device according to claim 4, wherein each of said membrane layers has a thickness of from about 0.125 mm to 0.250 mm.

6. A cell culture device according to claim 1, wherein said envelope has a longitudinal length relative to an external circumference of said core which allows for a plurality of layers of envelope to be spirally wrapped about said core.

7. A cell culture device according to claim 2, wherein said first delivery means and said first removal means comprise a plurality of capillaries having liquid permeable walls disposed within said envelope and open ends extending outwardly from between said sealed longitudinal edges of said envelope such that said capillaries communicate with said cell culturing space only through the walls of said capillaries.

8. A cell culture device according to claim 7, wherein said capillaries are disposed substantially equally spaced and substantially parallel to each other.

9. A cell culture device according to claim 8, wherein said capillaries are spaced from one another at a distance of from about 100 microns to about 1000 microns.

10. A cell culture device according to claim 1, wherein said second delivery means comprises at least one tube means disposed between said first longitudinal edges of said envelope and projecting into said cell culturing space such that a first end portion of said tube means communicates with said cell culturing space and a second end portion of said tube means communicates with an inlet means on said first end header means.

11. A cell culture device according to claim 7, wherein said core is gas permeable.

12. A cell culture device according to claim 11, wherein said core comprises a hollow material, the circumferential surface of which is perforated.

13. A cell culture device according to claim 11, wherein said third delivery means engages said core at a first end thereof so as to form a fluid-tight seal therewith thereby allowing gas to enter said core and which also communicates with said inlet means on said first end header means.

14. A cell culture device according to claim 11, wherein a second end of said core opposite a first end is sealed with a sealing means to prevent the escape of gas therefrom.

15. A cell culture device according to claim 11, wherein said third removal means comprises an opening defined by said second lateral edge of said envelope and said second external surface of a previously wrapped layer of said envelope through which gaseous waste products may exit said device.

16. A cell culture device according to claim 1, wherein said second removal means comprises at least one tubular means disposed between said second longitudinal edges of said envelope, and which projects into said cell culturing space such that a first end portion of said tube means communicates with said cell culturing space and a second end portion of said tube means communicates with said outlet means on said second end header means.

17. A cell culture device according to claim 1, wherein said adhesive means is further disposed along the entire length of said second lateral edge of said first external surface of said envelope such that said edge fixedly adheres to said second external surface of the previously wrapped layer of envelope.

18. A cell culture device according to claim 17, wherein said core is impermeable to gases.

19. A cell culture device according to claim 17, wherein said third delivery means comprises at least one tubular means disposed on and secured to said first external surface of said at least one envelope and which extends at least along a portion of said surface so as to be parallel with said longitudinal axis of said core and which also extends outwardly from said envelope to communicate with said inlet means on said first end header means.

20. A cell culture device according to claim 17, wherein said third removal means comprises at least one tubular means disposed on and secured to said first external surface of said at least one envelope and which extends along at least a portion of said surface and which also extends outwardly from said envelope so as to communicate with said outlet means on said second end header means.

21. A cell culture device according to claim 17, wherein said first delivery means and said first removal means comprises a plurality of capillaries disposed within said envelope and whose open ends extend outwardly from between said sealed longitudinal edges of said envelope such that said capillaries communicate with said cell culturing space only through the walls of said capillaries.

22. A cell culture device according to claim 21, wherein a plane defined by a surface of said core perpendicular to its longitudinal axis and said first longitudinal edges of said spirally wrapped envelope, and said first end header means together define a first plenum chamber which communicates with an open end of said capillaries.

23. A cell culture device according to claim 21, wherein a plane defined by a surface of said core perpendicular to its longitudinal axis and said second longitudinal edges of said spirally wrapped envelope, and said second end header means together define a second plenum chamber which communicates with an open end of said capillaries.

24. A cell culture device according to claim 1, wherein said device comprises two envelopes having substantially equal dimensions, wherein each of said two envelopes is formed by superimposing and bonding a gas-permeable hydrophobic membrane layer onto a semi-porous hydrophilic membrane layer along their first and second lateral and longitudinal edges, respectively, to define a cell culturing space therebetween, further provided that a first envelope is superimposed upon a second envelope such that said hydrophilic membrane layer of said first envelope faces said hydrophilic membrane layer of said second envelope and is bonded thereto along their first and second lateral edges thereof.

25. A cell culture device according to claim 24, wherein said hydrophilic membrane layer comprises regenerated cellulose acetate.

26. A cell culture device according to claim 24, wherein said first delivery means and said first removal means comprise a spirally extending channel defined between said two envelopes such that nutrient media is allowed to flow therethrough from between said first and second unsealed longitudinal edges thereof.

27. A cell culture device according to claim 24, wherein said third delivery means and said third removal means are disposed on and secured to an external surface of said gas-permeable hydrophobic membrane layer of one of said two envelopes.

28. A cell culturing device according to claim 1, wherein a mesh is placed atop at least one membrane layer of said at least one envelope.

29. A cell culturing device according to claim 28, wherein said mesh comprises a non-woven plastic screen.

30. A cell culturing device according to claim 1, wherein an outermost layer of spirally wrapped envelope is disposed within a shell means.

31. A cell culturing device according to claim 30, wherein said shell means comprises a heat shrinkable thermoplastic sleeve.

32. A cell culture device according to claim 11, wherein said device is disposed within a shell means which is substantially permeable to gases and/or perforated.